Bacillus anthracis is a bacterial pathogen that causes the often lethal disease anthrax. This research aims to characterize the role of potential virulence genes in Bacillus anthracis. Virulence is a pathogen’s ability to damage the host. Studying virulence allows us to understand infection mechanisms and develop novel ways to target pathogens. Previous work identified a collection of potential virulence mutants (Franks et al, 2014) each containing a genetic disruption that renders a gene non-functional. These mutants were pulled out in initial screenings but were never characterized further. We confirmed that one mutant, TN2, also exhibits decreased virulence in a Galleria mellonella survival assay. We know that TN2 has a disruption in a promoter region that we hypothesize controls two genes: a putative BNR repeat domain protein (TN2A) and a glycosyl-like 2 transferase family protein (TN2B). For my project, I attempted insertional mutagenesis to inactivate these genes with the goal of confirming that the genes are linked to virulence, rather than unintended mutations elsewhere in the genome. After successfully creating insertional mutant 2B, through the disruption of the TN2B gene, I am working to further characterize the mutant to determine its role in immune evasion. Specifically, I will compare the ability of the wild-type and mutants to survive exposure to various antimicrobial defenses conserved in humans and waxworms. This research could help identify a novel bacterial virulence factor and its potential mechanisms of action thus expanding our understanding of bacterial pathogenesis.

The gram-positive bacterium, Bacillus anthracis, is responsible for the deadly disease Anthrax. B. anthracis is dangerous due to virulence factors, or defenses the bacteria uses to infect a host. We hope to better understand how this bacterium interacts with its hosts by studying the genes necessary for virulence. Bacterial mutants, which have a change in their genetic sequence, sometimes show reduced ability to cause disease in a host. Studying these mutants helps us understand the bacteria’s infection method. Previously our lab created a library of mutants using a technique called transposon mutagenesis and then screened these transposon mutants for phenotypes linked to decreased virulence. This resulted in the identification of 11 transposon mutants that were less effective at causing disease in the nematode Caenorhabditis elegans (Franks et al.). While all 11 mutants could be interesting for further characterization, it is necessary to prioritize them as this is still too many to study. In this project, we tested these mutants using a second infection model, the caterpillar Galleria mellonella. G. mellonella is an ideal model due to its optimal size for injection, conserved innate immune defenses, and previous success as an infection model for B. anthracis (Malmquist et al.). We found that only one of these 11 mutants, TN2, had reduced virulence in both C. elegans and G. mellonella. Future research will focus on confirming the genetic change in this mutant and determining the mechanism by which it contributes to infection. This could lead to new antibiotic targets in the future.

Mercury (Hg) is released into the environment by coal-burning powerplants and artisanal gold mines. Aquatic bacteria then convert the inorganic mercury into highly toxic methyl mercury. Turtles acquire mercury through their diet, and it bioaccumulates throughout their long lifetime. Toenail clippings can be used to determine Hg concentrations in turtles. Toenail samples were collected from Trachemys scripta elegans (red-eared sliders) in the Brazos River near Granbury and the Clear Fork of the Trinity River as it flows through Fort Worth. All toenails were dried in a 60℃ oven and put into a direct Hg analyzer that uses thermal decomposition, gold amalgamation, and atomic absorption spectrometry to determine total Hg. Toenails from the Brazos river had significantly more Hg on average than those in the Clear Fork, 658.302µg/kg and 400.146µg/kg respectively. The results were unexpected as the Brazos river near Granbury is considered less polluted than the Clear Fork of the Trinity, which is supported by observations of insect larvae such as hellgrammites which were common in the Brazos but absent in the Clear Fork of the Trinity. Our hypothesis is that red-eared sliders in the Brazos are feeding at a higher trophic level than those in the Clear Fork. Fecal samples and a lack of invertebrate prey in the Clear Fork suggest red-eared sliders primarily feed on algae. In the Brazos river we observed several species of insect larvae underneath rocks and hypothesize that the red-eared sliders are feeding on this abundant food source. Mercury is known to biomagnify and therefore red-eared sliders in the Brazos are likely ingesting more mercury than those in the Clear Fork.

Alzheimer’s Disease (AD) and Traumatic Brain Injuries (TBI) are global societal problems affecting millions of people and costing billions of dollars per year.1,6 Hallmarks of AD include memory loss, cognitive decline, depression, and confusion due to unchecked inflammation in the brain caused by the overproduction of pro-inflammatory cytokines by the immune system.1,9,11 TBI occurs when a sudden trauma damages brain cells, which activate the immune response potentially leading to chronic inflammation and a multitude of symptoms affecting cognitive, somatic, and emotional processes.3,4,12 There is currently no cure for AD, nor is there an effective treatment for chronic inflammation caused by TBI. P2D Bioscience® has manufactured a series of drugs successfully targeting inflammation in a 3XTgAD mouse model.21 To understand the cellular mechanism of the novel drugs, we used SDS-PAGE electrophoresis and Western Blot analysis to investigate protein levels within the NFB pathway, which modulates inflammation. We monitored the inhibitor of NFb, IB, to determine whether the drugs were blocking the phosphorylation and degradation of IkBa and subsequently blocking the pro-inflammatory effects of activated NFB. We show that the drug is blocking the degradation of IB, and therefore the pro-inflammatory genes associated with the NFB pathway are not being transcribed. Increasing our understanding of the cellular mechanism of action is imperative for the progression of drug development because it can be used to evaluate potential side effects.

Alzheimer’s Disease (AD) is a neurodegenerative disease that primarily affects elderly populations. AD engenders memory loss and cognitive decline, and its prevalence is rapidly growing. It is estimated that 14 million Americans will have AD by the year 2050. Therefore, it is imperative for researchers to examine the underlying biological mechanisms responsible for AD. Previous research has demonstrated that chronic inflammation is linked to the hallmark AD pathology, amyloid beta (Aβ). Aβ is a protein that disrupts neuronal communication and increases the production of effector proteins called pro-inflammatory cytokines. Microglia function like immune cells in the brain, and when they are activated by inflammatory triggers, such as Aβ, they secrete pro-inflammatory cytokines. Although cytokine release is initially a healthy response, excess cytokine production is harmful to the brain and exacerbates AD pathologies. Prior research has demonstrated that pro-inflammatory cytokines are upregulated in the serum of AD patients. Therefore, cytokines are a crucial target for AD therapeutics.

The current experiment will examine the temporal inflammatory response of microglial cells following lipopolysaccharide (LPS) insult. LPS is a component of common bacteria and can induce inflammation in microglial cells. We will treat cells with several different concentrations of LPS and assess cytokine production at several different timepoints. To do this, we will collect cell supernatant (secretions) and measure multiple cytokines using an ultrasensitive electrochemiluminscent assay. Data collected from these experiments will be used in many future studies of potential therapeutics and dietary supplements. In fact, data from these experiments will be used by current and future departmental honors students to determine the optimal treatments and times for their experiments. This project is incredibly relevant because AD is currently the 6th leading cause of death in the United States. Data collected will help us pinpoint proper testing procedures for therapeutic compounds that are developed.